Nearly all of the known virulence factors synthesized by Bordetella pertussis, the causative agent of human whooping cough, and Bordetella bronchiseptica, which causes respiratory disease in other animals, are positively regulated by the Bvg signal transduction system. Bvg also negatively regulates production of a class of outer membrane proteins in B. pertussis and flagella synthesis and motility in B. bronchiseptica. The ability of Bordetella species to alternate between distinct phenotypic phases in response to environmental conditions has been recognized for many years, yet the role of Bvg- mediated signal transduction in the infectious cycle is unknown. The investigators' experiments will focus on the following questions: 1) How and why are Bordetella virulence genes regulated by Bvg, and 2) What are the functions of the regulated gene products? To address these they will use a comparative approach by conducting parallel studies with the human pathogen B. pertussis and the closely related animal pathogen B. bronchiseptica. They have established in vitro assays for studying the biochemistry of signal transduction, genetic methods for manipulating the virulence regulon, and natural host- animal models for characterizing respiratory tract infection. These form the foundation for this proposal. The research plan begins with an examination of molecular aspects of signal transduction using purified proteins and measurements of BvgA and BvgS function in E. coli. They will also address mechanisms of signal recognition by BvgS and these basic studies will facilitate an in vivo analysis. The effect of alterations in Bvg signal transduction pathways will then be assessed using animal models for B. bronchiseptica and B. pertussis. Studies with defined mutants will be accompanied by the development of a method for directly determining the phase of Bordetella populations in respiratory tissue. The relationship between the virulence regulons of B. pertussis and B. bronchiseptica will be investigated next, with specific emphasis on loci that are negatively regulated by Bvg. Finally, they will assess the functions of putative adhesins and toxins during infection of natural hosts by B. bronchiseptica. A novel approach using "ectopic expression" will be used for this purpose. By considering the Bvg regulon from a broad perspective they hope to discover its role in pathogenesis.